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    Analytical Biochemistry, submitted 02/16/2010

    1

    PASS: A High Throughput Assay for Screening and

    Selection of Detergents for Membrane Proteins

    James M. Vergis1, 2 and Michael C. Wiener1, 3

    1Department of Molecular Physiology and Biological Physics, University of Virginia, 480 Ray

    C. Hunt Drive, Charlottesville, VA 22908.

    2Current address: Department of Microbiology and Immunology, Uniformed Services

    University of the Health Sciences, Bethesda, MD 20814

    3Correspondence should be addressed to M.C.W. ([email protected]); 434-243-2731; 434-

    243-8271 (FAX).

    Short Title: Membrane Protein Detergent Stability Assay

    Category: Membranes and Receptors

    Keywords: Membrane protein detergent stability, high throughput assay, pre-crystallization

    screening, structural biology.

    Total Pages: 24

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    Abstract

    Structural studies on integral membrane proteins are routinely performed on protein-detergent

    complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all of

    the membrane proteins crystal structures solved to date, a subset of only four detergents has been

    used in over half of these structures. Unfortunately, many membrane proteins are not well-

    behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of

    detergents that maintain the solubility and stability of a membrane protein is a critical step, and

    can be a lengthy and protein-expensive process. We have developed an assay that characterizes

    the stability and size of membrane proteins exchanged into a panel of 94 commercially available

    and chemically diverse detergents. This Prompt Assay of Stability and Size (PASS), utilizing a

    set of SBS-format filtered microplates, requires sub-milligram quantities of purified protein,

    small quantities of detergents and other reagents, and is performed in its entirety in several hours.

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    1 Abbreviations used: B22, second virial coefficient; CMC, critical micelle concentration; CV,

    column volume; DM, n-decyl--D-maltopyranoside; FSEC, fluorescence-detection size-

    exclusion chromatography; GHP, GH polypro hydrophobic polypropylene; IMAC, immobilized

    metal ion affinity chromatography; LDAO, n-dodecyl-N,N-dimethylamine-N-oxide; MW,

    molecular weight; MWCO, molecular weight cut-off; OG, n-octyl--D-glucopyranoside; PASS,

    prompt assay of stability and size; PBS, phosphate buffered saline; PDC, protein-detergent

    complex; PCR, polymerase chain reaction; PES, polyethersulfone; SBS, Society for

    Biomolecular Sciences; SEC-M, size-exclusion chromatography mimesis; TCA, trichloroacetic

    acid.

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    The difficulties of working with membrane proteins are demonstrated by the fact that membrane

    protein structures represent less than 1% of the total number of protein structures in the Protein

    Data Bank, despite integral membrane proteins encompassing 15-30% of most genomes [1; 2; 3].

    Technical challenges in membrane protein structure determination include expression (to obtain

    suitable amounts of protein), purification (to obtain suitably stable and functional protein), and

    sample preparation (to obtain suitable two-dimensional crystals [for electron crystallography],

    three-dimensional crystals [for x-ray crystallography], or solutions [for NMR spectroscopy]. The

    assay technology developed and presented here focuses upon the selection of appropriate

    detergents for use with a specific membrane protein, which is a critical aspect of both

    purification and sample preparation. In preparation for structural (and other) studies, membrane

    proteins are extracted from their native lipid bilayer environment, and this membrane bilayer is

    replaced by a membrane-mimetic. The membrane-mimetic solute is almost always a detergent at

    a concentration above its critical micelle concentration (CMC)1, where the detergent surrounds

    the hydrophobic membrane-facing portion of the membrane protein and forms the protein-

    detergent complex (PDC). PDCs are in equilibrium with detergent micelles and monomers in this

    solution. The chemical-space of detergents is large, and the solution (and crystallization)

    properties of a membrane protein are intimately related to the properties of the detergent(s)

    comprising the PDC [4; 5]. Also, the function of a membrane protein can be maintained at native

    or near-native levels or can be completely abrogated, depending upon the detergent composition

    of the PDC.

    Currently, according to the Membrane Proteins of Known Structure database

    (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html) , 231 unique integral membrane

    http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.htmlhttp://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
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    protein structures have been solved by x-ray crystallography. The Membrane Protein Data Bank

    (http://www.mpdb.ul.ie/) [6] lists 864 non-unique membrane protein x-ray crystal structures, for

    which more than fifty different detergents have been used in their solubilization and/or

    crystallization. These detergents are not equally represented. For example, five detergents, n-

    dodecyl--D-maltopyranoside (DDM); n-decyl--D-maltopyranoside (DM); n-nonyl--D-

    glucopyranoside (NG); n-octyl--D-glucopyranoside (OG); and n-dodecyl-N,N-dimethylamine-

    N-oxide (LDAO) have yielded the majority of-helical membrane protein structures [7]. While

    this speaks to the utility (and extensive use) of these five detergents, over 40% of the structures

    solved to date required detergents other than those five. As such, survey of membrane protein

    stability in detergent-space is an important aspect of membrane protein structural biology (and

    biochemistry).

    There are several methods to test detergent solubility of membrane proteins. These methods

    include: gel filtration; dilution [5]; and the ultracentrifugation dispersity sedimentation assay [8].

    Inspection of the gel filtration chromatogram has been routinely used for both soluble and

    membrane proteins to assess the quality of a protein. The method of fluorescence-detection size-

    exclusion chromatography (FSEC) is a major advance in gel filtration chromatography of

    integral membrane proteins [9]. The unique optical signal of a fluorescently-tagged recombinant

    protein enables that protein to be detected and characterized in a solubilized mixture, prior to

    purification. Also, the use of fluorescence (versus absorbance) detection increases the sensitivity

    by several orders of magnitude, requiring less solubilized (or purified) protein for the

    chromatography analysis. In order to evaluate detergent stability, gel filtration can be performed

    in either of two ways: 1) the column is equilibrated in the detergent to be tested and the protein is

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    loaded onto the column (detergent-specific mobile phase) or 2) the protein is exchanged into a

    new detergent and then injected onto a column equilibrated with a known good detergent for

    all chromatographic runs (generic mobile phase). The use of the generic mobile phase speeds

    up the gel filtration runs by eliminating the column washing and equilibration steps for the next

    detergent. The generic mobile phase method rests upon the assumption that if a protein sample

    has been exchanged into an incompatible detergent, then a compatible detergent in the mobile

    phase will not reverse the deleterious effects of that incompatible detergent [9]. Data from our

    lab suggests that this is not true for all cases, so we do not currently favor the generic mobile

    phase method. We note that the original FSEC publication [9] utilizes a generic mobile phase;

    however, fluorescence detection is equally applicable to use of a detergent-specific mobile phase.

    For the dilution method, concentrated protein is diluted into a new test detergent and the

    Abs320nm:Abs280nm ratio is recorded over time. Since Abs320nm is indicative of protein

    aggregation, an increase in this ratio is diagnostic of the protein not being stable in the new

    detergent [5]. In the ultracentrifugation dispersity sedimentation assay, the protein is

    concentrated, diluted into the test detergent buffer with three concentration/dilution steps, and

    finally allowed to incubate overnight. At this point a sample is taken while the rest of the protein

    is spun in the ultracentrifuge to pellet any aggregated protein. Another protein sample is taken

    after ultracentrifugation and both the pre- and post-ultracentrifugation samples are run on SDS-

    PAGE and compared. Any difference in band intensity between the two samples is indicative of

    aggregated protein being removed during the intermediate ultracentrifugation step and thus

    related to detergent stability [8].

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    These three methods all possess shortcomings. The biggest limitation is that the methods

    described above are not detergent exchanges, but rather are detergent dilutions (the exception is

    the single case where the protein is already in the same detergent as that present in the gel

    filtration mobile phase). This is a problem if the initial detergent is not diluted to a concentration

    below its CMC or, in the case of gel filtration, if the original detergents micelles are not

    separated from the protein-detergent complex (PDC), or if a mixed detergent population exists.

    In these instances, the presence of the original detergent can protect a protein from a

    destabilizing detergent resulting in false positives. The original detergents concentration is of

    great concern especially when the method utilizes a protein ultrafiltration concentration step

    since detergent micelles typically concentrate along with the protein even when a large

    molecular weight cut-off (MWCO) is used. Another limitation is that milligram amounts of

    protein and large amounts of expensive detergent reagents may be necessary, especially if there

    are a large number of conditions to be tested. Lastly, the time required to perform each method

    can be long, which usually limits the number of detergents surveyed, especially in the case of gel

    filtration where only one detergent can be tested at a time.

    We have developed a microplate-based detergent screening assay called PASS (Prompt Assay of

    Stability and Size) (Fig. 1). A standard buffer exchange technique, successfully implemented in

    screening detergents suitable for extracting proteins from their membrane environment [10; 11;

    12], is used to exchange microgram quantities of purified membrane protein into each of the

    detergents of our 94 detergent panel (Table 1). This simple technique involves binding the

    protein to an affinity resin, extensively washing with the new detergent, and finally eluting from

    the column in the new detergent. In parallel, these eluents are each passed through high and low

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    molecular weight cutoff (MWCO) filter plates. The amounts of protein in the filtrates are

    measured by a rapid Western-blot protocol. PASS is performed with 96-well SBS format filter

    plates, for further increasing speed and decreasing reagent and protein costs. Our assay addresses

    the limitations of the detergent screening methods described above, and provides measures of

    both stability and rudimentary size information. Typically, results can be obtained in

    approximately two hours with only few hundred micrograms of protein required for the assay.

    Furthermore PASS can be completely automated if desired. To validate the assay, we present

    PASS data of two membrane proteins, AqpZ and KcsA. Both of these membrane proteins have

    been crystallized and their structures initially solved by other laboratories.

    For the purpose of this manuscript (and corresponding to a standard empirical definition), we

    define the stability of a membrane protein in a given detergent to be a quantity that is inversely

    proportional to the fraction of PDCs that form large particles or aggregates in that specific

    detergent. If all of the protein forms large particles or aggregates (which would be present in the

    void volume of a suitable gel filtration column, for example), then we would call that protein

    sample unstable. If none of the protein forms large particles or aggregates (such that the protein

    would be seen as one or more sizing peaks in the aforementioned gel filtration column), then we

    would call that protein sample stable. [Of course, stability, generally a time-dependent property,

    is not affected solely by detergent.]

    Materials and Methods

    Detergent panel

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    All chemicals for the detergent panel were purchased from Anatrace, Avanti Polar Lipids,

    Bachem, and EMD Biosciences as indicated in Table 1. 2X working stock solutions were made

    in ultra-pure water, dispensed into 96-well plates, heat sealed with foil tape and frozen at -20C

    until needed.

    MWCO filtered microplate assessment

    Stock solutions of each gel filtration MW standard (Sigma and GE Healthcare) at 2-5mg/ml were

    made in PBS buffer. 30l of each stock solution was added to a 0.2m filter plate (Part #5045,

    Pall Corp.) and 100kDa and 300kDa MWCO filter plates (#T-3180-14 and #T-3180-21 ISC

    Bioexpress, or #CMR1411 and #CMR1493-1 Seahorse Labware) and spun at 2000xg for 2 min.

    We emphasize that these values of 100kDa and 300kDa MWCO are the names given to these

    plates by the manufacturer. As will be shown, the actual MWCOs measured are different. We

    will henceforth use the nomenclature LOW and HIGH for the 100kDa and 300kDa plates,

    respectively. The filter plate flow through along with samples of the original stock solutions

    were transferred to a UV transparent 384-well plate and Abs280nm was measured on a Molecular

    Devices SpectraMax 384 Plus spectrophotomer with PathCheck (i.e. 1cm path length correction)

    active. The percent difference between the stocks and eluate absorbance values were used to

    calculate the flow through percent for each standard through each filter type. The errors are the

    standard deviations obtained from measuring three separate samples in each filtered microplate.

    Purification of AqpZ and KcsA

    Both (His)6-AqpZ (cys-free) and (His)6-KcsA were over-expressed and purified fromE. coli by

    slight modification of published methods [13; 14]. The AqpZ buffer was 20mM Tris pH 7.4,

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    500mM NaCl, 10% glycerol, and 40mM OG. The KcsA buffer was 20mM Tris pH 7.4, 150mM

    KCl, and 1mM DDM. Both purifications were only carried out to the first IMAC step with Co-

    TALON (Clonetech) resin and then desalted back into the respective buffers with PD10 columns

    (GE Healthcare). KcsA was further processed by digestion with chymotrypsin (Worthington

    Biochemical Corp.) for 4hrs at RT with a 1:25 Chymotrypsin:KcsA ratio by mass. The

    chymotrypsin was removed with immobilized benzamidine (GE Healthcare).

    Detergent stability assay

    400g of AqpZ or KcsA were batch-bound to 1.5ml of Co-TALON resin (0.27g protein/l

    resin) along with additional buffer to a final volume of 7.5ml making a 20% slurry. 50l of the

    protein-bound resin slurry (10l resin) was then added to the 0.2m filter microplate using a

    multichannel pipette with the ends of the tips cut to make a wider bore. The resin was pelleted in

    the microplate by spinning at 2000xg for 2min at 4C. The detergent containing wash solutions

    were prepared from the 2X detergent stock panel and 2X buffer stocks. 3CV (Column Volumes)

    of the detergent wash solution was added to each well and the microplate was spun at 2000xg for

    2min at 4C to remove the buffer. This washing is repeated until 20CV of detergent wash buffer

    had been added. The elution solutions were prepared from the 2X detergent stock panel and 2X

    elution buffer stocks (2X wash buffer containing 1M imidazole). The detergent exchanged

    protein was then eluted from the resin in 6CV of elution solution by spinning the plate at 2000xg

    for 2min at 4C into a PCR collection plate (Abgene). 25l of the eluted protein was then added

    to the 300kDa (i.e. HIGH) MWCO filter microplate and 25l added to the 100kDa (i.e. LOW)

    MWCO filter plate, the plates spun at 2000xg for 2min at 4C and the eluate collected in a PCR

    collection plate. The Minifold I 96-well Dot-Blot apparatus (Whatman) was used to blot the

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    elution samples onto 0.2m nitrocellulose (Whatman). Due to the spot-broadening effects of

    some detergents, the protein was precipitated in the blotting apparatus with trichloroacetic acid

    (TCA) prior to applying the vacuum. 150l of 10% TCA was first added to each well of the

    assembled dot-blot apparatus followed by 10l of the eluate from each MWCO filter plate.

    Vacuum was applied to filter the sample through the nitrocellulose and each well was washed

    with 20mM Tris pH 7.4, 500mM NaCl three to four times. The membranes were quickly

    washed with ultra-pure water and blocked for 10min with Odyssey blocking buffer (LI-COR

    Biosciences). The membranes were probed for 10min with an IRDye800CW conjugated anti-

    6X His Tag antibody (Rockland Immunochemicals) diluted 1:10000 in Western Breeze Primary

    Antibody Diluent (Invitrogen). The blots were washed with Western Breeze Antibody Wash

    (Invitrogen) four times for 2min each and then with ultra-pure water. The blots were scanned on

    an Odyssey Infrared Imaging System (LI-COR Biosciences) with the intensity adjusted to avoid

    saturation of the spots. Integrated spot intensities were measured with the Odyssey software and

    the background for each spot was calculated from the median value of the baseline surrounding

    the spot. The background-corrected integrated spot intensities were then exported to a

    spreadsheet for normalization and graphing.

    Gel filtration runs

    Detergent exchanges using larger amounts of protein (12g protein/l resin) for gel filtration

    were carried out in 0.22m filter spin columns (Millipore) and a table top centrifuge using the

    same detergent exchange protocol above except the protein was eluted in only 3CV and 150mM

    EDTA was substituted for imidazole in the elution buffer in order to avoid imidazole

    fluorescence during gel filtration. 10l eluted protein was loaded onto a calibrated Superdex

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    200 5/150 GL short column (GE Healthcare) at 0.4ml/min. The column was equilibrated in

    the exchange detergent prior to sample injection. The intrinsic protein fluorescence

    (Ex280nm/Em335nm) was monitored using a Hitachi L-2485 fluorescence detector.

    Results

    Size-exclusion chromatography mimesis (SEC-M)

    The use of multiple differing molecular weight cutoff (MWCO) filters in parallel permits

    acquisition of rudimentary size information on PDCs. It is important to note that MWCO filters

    do not have a single sharp molecular weight cutoff. Instead, a given filter will exclude

    approximately all particles above a certain size, will pass approximately all particles below a

    certain size, and will let through some fraction of particles between these two limits. For the

    purposes of PASS, consider two MWCO filter plates: HIGH and LOW. The HIGH plate

    excludes particles of large size and permits some or all of the remaining particles to pass

    through. The retentate of the HIGH plate will consist solely of these large particles; nearly

    everything else will be in the filtrate. The LOW plate has a lower size-exclusion limit. Therefore,

    the retentate of the LOW plate will include all of the retentate present in the HIGH plate plus

    additional retentate arising from the lower cutoff. Thus, in the absence of experimental error, the

    fraction of a sample in the filtrate of the LOW plate will always be less than or equal to the

    fraction of that sample in the HIGH plate filtrate.

    During the conception of the detergent screening assay, gel filtration of the eluted samples was

    planned to be performed using either a generic mobile phase or detergent-specific mobile phase

    in conjunction with an in-line fluorescence detector to follow intrinsic protein fluorescence of the

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    protein of interest. It became readily apparent that this would not work due to the fluorescence

    of a large population of the detergents in the panel themselves (data not shown). As an

    alternative to gel filtration for obtaining sizing information, or at the very least to remove any

    protein aggregate analogous to that present in the the void volume of a gel filtration run,

    filtration through MWCO PES (polyethersulfone) filters was employed. Since MWCO PES

    filters are not absolute cut-offs but instead permit a range of particle sizes to pass through their

    membrane, SBS format MWCO filterplates from several manufactures were evaluated. The size

    distribution range for each MWCO filterplate was measured using gel filtration MW standards.

    A sizing microplate was sought that would disallow passage of blue dextran (the 2000kDa void

    volume MW standard) and produce a suitable MW distribution for estimating size. We

    emphasize that a single MWCO filtered microplate was not sufficient to satisfy both criteria

    since the MW permeability range was too narrow for the LOW plate and too broad for the HIGH

    plate. To overcome this deficiency, LOW and HIGH MWCO microplates were paired together

    for the assay. The MW distributions of these two MWCO filtered microplates along with the

    0.2m GHP (GH Polypro, hydrophobic polypropylene) filter plate are shown in Fig. 2. As

    expected, the 0.2m plate allows passage of all of the gel filtration standards, with equal and

    complete permeability of all of standards except blue dextran (partially permeable). In contrast,

    neither the LOW nor the HIGH plates allow blue dextran to flow through, and thus prevent

    highly aggregated protein from passing through. With the use of both MWCO microplates,

    analysis of the HIGH microplate filtrate reports primarily on stability, and the ratio of the LOW

    and HIGH plate filtrates reports on size. Thus, MWCO filters are used to mimic sizing data

    obtained sizing data obtained by gel filtration chromatography. We call this technique Size-

    exclusion Chromatography Mimesis or SEC-M.

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    The detergent panel

    The assay in its current form utilizes 96-well SBS format microplates. We utilize our 94-

    detergent panel (Table 1) plus negative and positive positions A1 and A2 of the microplates. The

    negative control contains detergent free buffer, which will cause the protein to precipitate on the

    resin, while the positive control is the current detergent containing buffer. Even though it is

    highly probable that the current detergent used for a given protein is present in the panel, the

    working concentrations of the detergent may be different. The chemical (or trademarked

    names), well locations, abbreviations, working concentrations, and CMC values for each

    detergent in the panel are shown in Table 1. All of the detergents were selected for their

    suitability for membrane protein studies based upon several criteria: 1) commercial availability;

    2) moderate or high aqueous solubility; 3) CMC values between 0.03 and 40mM; and 4)

    zwitterionic or nonionic headgroups. The detergent panel is first grouped into zwitterionic and

    nonionic sets, further ordered by chemical class, then by chain length within each chemical class

    where applicable, and finally by CMC value. This permits the facile recognition of patterns of

    stability. The detergent stock plate consists of 2X working concentration solutions dispensed and

    heat sealed into single use microplates, for ease in formulation of the wash and elution buffers.

    Each detergents working concentration in the panel varies with respect to its CMC value: 2X

    (CMC > 10mM); 2.5X (CMC 5-10mM); 3X (CMC 1-5mM); 10X (CMC 0.5-1mM); 50X (CMC

    0.1-0.5mM); 100X (CMC < 0.5mM). The exception is TDM where the working concentration is

    50X due to solubility issues. The use of several CMC multiples is necessary due to low-CMC

    detergents being required at higher concentrations (in terms of their CMC) than high-CMC

    detergents [5].

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    Visualization and quantification of the elutions

    Using a fast Western protocol, the elutions for each MWCO microplate are blotted to a

    nitrocellulose membrane, visualized using a conjugated primary antibody specific to the affinity

    tag used, and quantified in approximately one hour. Dot blots of the elutions from the AqpZ and

    KcsA detergent exchanges are shown in Fig. 3. Quantification of dots on the blots measures the

    amount of protein eluted from each well. Since the same amount of protein was used in each

    well and an incompatible detergent will cause a membrane protein to precipitate on the affinity

    resin, the integrated dot intensity is directly related to the ability of a particular detergent to

    stabilize a membrane protein relative to the other detergents in the panel. As mentioned in the

    introduction, the loss of membrane protein stability is assayed by the presence of and amount of

    irreversible aggregates (large particle formation) as a function of the detergent species that is

    present in the PDC. The LOW and HIGH dot blots are quantified and normalized to the highest

    intensity dot on each respective blot. The ratios of the normalized LOW to HIGH dot intensities

    are calculated to indicate the relative size of the PDC; this ratio is inversely proportional to the

    PDC size, with larger ratio values indicative of a smaller PDC size. The ratio is a better estimate

    of size than just the LOW dot intensities alone. The LOW dot intensities are related to both size

    and stability, while the LOW/HIGH ratio accounts for the removal of any aggregated protein

    retained by the HIGH microplate (and thus also retained by the LOW microplate). Fig. 4and

    Fig. 5 demonstrate two representations of the quantified data. Fig. 4, the audio equalizer

    representation, presents the data to allow any stability and/or size patterns related to detergent

    type, chain length, or CMC to be easily elucidated since the detergent panel is organized in that

    manner. Fig. 5, the size-stability quad- plot, displays the data to allow quick assessment of

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    PDC size related to stability without any regard for the detergents physical or chemical

    properties. The data in both representations are binned into quartiles to help compensate for the

    inherent error in measuring only one data point. Interestingly, despite AqpZ being a larger

    tetramer (103kDa), the KcsA tetramer (55kDa) possesses a much more variable size dependence

    as demonstrated by the broader distribution of relative PDC size seen in Fig. 5.

    SEC-M correlates with gel filtration

    In order to further validate the use of the SEC-M technique in the assay, larger scale detergent

    exchanges were performed on AqpZ and KcsA and samples were run over a calibrated gel

    filtration column. The retention times of the gel filtration runs were compared to the dot

    intensities obtained from the two MWCO filter plate elutions. Fig. 6 (top panel) shows three

    AqpZ gel filtration runs exchanged into TDM, LDAO, and OG along with the corresponding dot

    blots for those detergents. As predicted from the LOW elution dot blots, the apparent PDC size

    order of AqpZ is OG < LDAO < TDM which is inversely proportional to their respective dot blot

    intensities. Fig. 6 (bottompanel)demonstrates an interesting phenomenon in which the same

    detergent at two different concentrations gives two different PDC sizes for KcsA. KcsA in the

    traditional working concentration of 1mM DDM is predicted to have a larger PDC size than the

    same protein in 8.5mM DDM by both gel filtration and SEC-M. The reason for a higher

    concentration of detergent reducing the size of the PDC is currently under investigation.

    Discussion

    The detergent panel presented here, when coupled to PASS, provides a robust and rapid means to

    survey membrane protein stability in a large number of chemically diverse detergents as well as

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    to obtain rudimentary PDC sizing information from SEC-M. While the sizing information

    obtained from SEC-M does not provide an absolute value for apparent MW, it does provide a

    coarse filter that allows the results to be binned into sizing and stability quartiles and ranked for

    further analysis via traditional gel filtration as desired. The ability to obtain quickly this sizing

    and stability information is a significant benefit to this method, especially if the time that would

    be required to examine all 94 samples with gel filtration is considered. For the Superdex 200

    5/150 GL column used in this work, each run is approximately 6 minutes in duration. Use of a

    generic mobile phase would require approximately 10hrs, and use of detergent-specific mobile

    phases would require an additional 44hrs for column equilibration and pump washing. These

    time estimates assume no down time and complete automation of the chromatography.

    Furthermore, many protein samples will aggregate or denature during this time, leading to

    ambiguous results.

    Upon inspection of Fig. 4, various detergent stability and size trends are seen for AqpZ and

    KcsA. We present several examples of the types of observations that can be obtained from

    PASS. For AqpZ, there is increased stability with an increase in detergent chain length seen with

    dimethylamine-N-oxide detergents (A7-A9), while decreased stability is observed with

    increasing ethylene chain length in the C8En detergents (E2-E4). For KcsA, increasing

    detergent chain length for glucoside detergents (F5-F7) resulted in decreased stability while just

    the addition of a hydroxyl group on Big CHAP (E12) forming Big CHAP, deoxy (F1) increases

    protein stability.

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    As mentioned in Results, Fig. 6shows an interesting phenomenon in which the smaller KcsA

    tetramer (55kDa) has a broader size distribution than that of the larger AqpZ tetramer (103kDa).

    This is most likely related to the amount of detergent KcsA binds relative to AqpZ to maintain its

    solubility (i.e. the more bound detergent, the larger the PDC size). The observation that a higher

    concentration of DDM resulted in smaller PDC size for KcsA (Fig. 6, bottom panel) suggests

    that more detergent is required to keep KcsA in a more compact state. Since the DDM monomer

    concentration should be relatively constant and approximately equal to the CMC at both 1mM

    and 8.5mM DDM concentrations [15], a higher number of DDM micelles appears to be required

    to form a smaller PDC. Experiments, planned to investigate this phenomenon, include

    determination of the amount of DDM bound to KcsA as a function of DDM concentration. The

    amount of detergent present in the PDC can be determined by static light-scattering coupled with

    refractive index and UV detection [16; 17; 18; 19]. In the context of the results observed with

    KcsA, we note that, contrary to the most simple expectations, detergent micelle size can change

    as a function of detergent concentration [20; 21].

    The readout from the size-stability quad-plot (e.g., Fig. 5)may help predict which detergents are

    best for crystallization since each quadrant represents different levels of stability and PDC size.

    Future experiments are planned to determine if a crystallization-quadrant exists. This would

    be somewhat analogous to the second virial coefficient (B22) crystallization slot where it was

    demonstrated that soluble proteins with B22 values within a specific narrow range had a

    propensity to form crystals [22]. Similar observations have been extended to membrane proteins

    [23; 24; 25; 26]. While detergents in the smallest, most stable quadrant would likely be useful

    for NMR, there is not enough data at present to suggest that this is necessarily the best criteria

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    19

    for membrane protein crystallography. Whether or not a crystallization quadrant exists, our

    assay and detergent panel will help with the critical pipeline step of choosing the proper

    detergents for any membrane protein of interest quickly and with minimal quantities of reagents.

    The two test proteins, AqpZ and KcsA, used in this study have both had their structures

    determined [13; 14; 27]. What is the location in the size-stability quad-plot for the

    crystallization detergents of AqpZ and KcsA? AqpZ was crystallized in n-octyl--D-

    glucopyranoside (OG) [14; 28]. OG [F6, Table 1] is located in the second-highest stability and

    second-smallest size quadrant (Fig. 5). For KcsA, Doyle et al. report purification of KcsA in n-

    decyl--D-maltopyranoside (DM) followed by detergent exchange via dialysis into n-dodecyl-

    N,N-dimethylamine-N-oxide (LDAO) [13]. DM [G10, Table 1] is located in the second-highest

    stability and second-smallest size quadrant (Fig. 5). LDAO [A9, Table 1] is located in the third-

    highest (second-lowest) stability and smallest size quadrant (Fig. 5). As the degree of exchange

    is not stated, we cannot unambiguously assign accurately the crystallization detergent (or

    detergent mixture) of KcsA. However, it is interesting to note that crystal structures of KcsA:Fab

    complexes (e.g.[29]) have essentially all been in DM. Based upon these scant examples, it is

    tempting to speculate that crystallization may be best pursued from the upper-left quarter of

    the quad-plot (i.e. those detergents that lay within the more stable and smaller halves of the

    distribution), but such conclusions await further data.

    With structural biology methodologies moving towards performing experiments on smaller

    scales with smaller amounts of material (especially important for difficult research problems

    with limited and/or expensive reagents), the development and use of high throughput

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    methodologies have increased. We present here a true high throughput membrane protein

    detergent screening assay that can be completed in approximately two hours with microgram

    amounts of protein and microliter volumes of reagents. PASS helps to overcome the barrier of

    low protein yields which are unfortunately typical for membrane proteins, especially when using

    more complex and/or higher order expression systems (e.g. eukaryotic). The assay as presented

    here used 400g of protein to obtain stability and PDC sizing information on 94 different

    detergents from our panel. This amount of protein is not the absolute minimum amount required

    to perform the assay. PASS could be conducted with 10-50g of protein (or even less) if more

    sensitive detection of the LOW and HIGH MWCO elutions could be performed. One approach

    to increase sensitivity is to probe the dot blot Western with primary and secondary antibodies

    that will amplify the signal at least several-fold. Alternatively, the use of radio-labeled or

    fluorescently-labeled protein could potentially increase sensitivity by several orders of

    magnitude or more. Lastly, while we have focused solely upon the use of PASS for the parallel

    screening of multiple single detergents, PASS can readily be extended to the screening of

    detergent mixtures, additives, ionic strength, pH and any other variable buffer components for

    both membrane and soluble proteins.

    Acknowledgements

    Funding for this research was provided by NIH Roadmap Grant 5R01 GM075931 (to M.C.W.).

    We thank Profs. Salem Faham and Jochen Zimmer for critical reading and discussion of the

    manuscript.

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    FIGURE AND TABLE LEGENDS:

    Fig. 1 Flowchart of the detergent stability assay. The generic protocol for performing the assay

    is presented.

    Fig. 2 Filter plate flow through of molecular weight standards. The Abs280nm was measured for

    each stock solution, and the eluate from each plate in triplicate. The percent difference between

    the elutate and the stock is presented on the graph. The error bars represent the error propagated

    from the standard deviation of each triplicate measurement. No passage of blue dextran was

    observed in either the HIGH or LOW MWCO filter plates.

    Fig. 3 Dot blots of eluted protein after exchange into the new detergent. 10l of the elutions from

    the HIGH and LOW MWCO plates were spotted on nitrocellulose membrane and visualized by

    Western blot using an IRDye800CW conjugated His-tag antibody and a LI-COR Odyssey

    imaging system. The dot intensities were quantified with median background correction within

    the Odyssey software.

    Fig. 4 Stability and relative size bar graphs. The normalized intensities from the HIGH MWCO

    dot blots are plotted along with the ratio of LOW:HIGH (LOW/HIGH) normalized intensities for

    AqpZ and KcsA. The values are grouped into quartiles, indicated by the gridlines. The HIGH

    intensity is directly proportional to stability while LOW/HIGH is inversely proportional to the

    particle size. Non-real ratio values (i.e. LOW intensity greater than HIGH intensity) are given in

    parenthesizes. These non-real ratio values are all from HIGH and LOW intensities within the

    same quartile rank except those indicated by an asterisk (*).

    Fig. 5 Quartile grid plot. The normalized intensities from the HIGH MWCO dot blots are

    plotted on the horizontal axis while the ratios of LOW:HIGH (LOW/HIGH) normalized

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    intensities for AqpZ and KcsA are plotted on the vertical axis. The well numbers are shown next

    to each dot. Non-real ratio values (i.e. LOW intensity greater than HIGH intensity) are located

    in the grayed out area of the plot. These non-real ratio values are all from HIGH and LOW

    intensities within the same quartile rank except those indicated by an asterisk (*).

    Fig. 6 LOW MWCO elution intensity correlates to HPSEC retention time. Larger amounts of

    AqpZ and KcsA were detergent exchanged using spin columns and then 10l injected onto a

    calibrated Superdex 200 5/150 GL gel filtration column equilibrated in the exchanged buffer.

    The retention times for MW gel filtration standards are shown on each chromatogram. The

    insets show the dot blot spots for each detergent.

    Table 1 Detergent screen.Note. The components in the detergent plate are shown above along

    with their locations in the plate and concentrations used. The values in parenthesis in the [Det]

    column are the CMC values for each detergent. Detergents in bold were purchased from Avanti

    Polar Lipids, italics from Bachem, underlined from EMD Biosciences, and all others from

    Anatrace. Detergents A3 through C9 are zwitterionic while C10 through H12 are nonionic

    detergents.

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    Table 1 Detergent ScreenWell Abbrev. Name [Det] mM Well Abbrev. Name [Det]

    A1 No detergent (- control) E1 C7E5 Pentaethylene glycol monoheptyl ether 42 (2

    A2 Empty well for current detergent (+ control) E2 C8E4 Tetraethylene glycol monooctyl ether 20 (

    A3 Z3-12 ZWITTERGENT

    3-12 8.4 (2.8) E3 C8E5 Pentaethylene glycol monooctyl ether 17.75

    A4 Z3-14 ZWITTERGENT

    3-14 10 (0.2) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (1

    A5 DMG n-Decyl-N,N-dimethylglycine 38 (19) E5 C10E5 Pentaethylene glycol monodecyl ether 8.1 (0

    A6 DOMG n-Dodecyl-N,N-dimethylglycine 4.5 (1.5) E6 C10E6 Hexaethylene glycol monodecyl ether 9 (0

    A7 DAO n-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) E7 C10E9 Polyoxyethylene(9)decyl ether 3.9 (

    A8 UDAO n-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) E8 C12E8 Octaethylene glycol monododecyl ether 9 (0.

    A9 LDAO n-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5 (0.

    A10 C-DDFOS C-DODECAFOS 44 (22) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0

    A11 CF-4 CYCLOFOS-4 28 (14) E11 C13E8 Polyoxyethylene(8)tridecyl ether 10 (0

    A12 CF-5 CYCLOFOS-5 13.5 (4.5) E12 CHAP Big CHAP 8.7 (2

    B1 CF-6 CYCLOFOS-6 8.04 (2.68) F1 CHAP-D Big CHAP, deoxy 4.2 (

    B2 CF-7 CYCLOFOS-7 6.2 (0.62) F2 OHES Octyl-2-hydroxyethyl-sulfoxide 48.4 (2

    B3 FC-10 FOS-CHOLINE-10 22 (11) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (2

    B4 FC-11 FOS-CHOLINE-11 5.55 (1.85) F4 GX-100 Genapol

    X-100 7.5 (0

    B5 FC-12 FOS-CHOLINE-12 4.5 (1.5) F5 HTG n-Heptyl--D-thioglucopyranoside 58 (2

    B6 FC-13 FOS-CHOLINE-13 7.5 (0.75) F6 OG n-Octyl--D-glucopyranoside 36 (1

    B7 FC-14 FOS-CHOLINE-14 6 (0.12) F7 NG n-Nonyl--D-glucopyranoside 16.25

    B8 FC-I11 FOS-CHOLINE-ISO-11 53.2 (26.6) F8 CYGLU-3 CYGLU

    -3 56 (2

    B9 FC-I11-6U FOS-CHOLINE-ISO-11-6U 51.6 (25.8) F9 HECAMEG HECAMEG 39 (1

    B10 FC-I9 FOS-CHOLINE-ISO-9 64 (32) F10 HEGA-9 Hega

    -9 78 (3

    B11 FC-U10-11 FOS-CHOLINE-UNSAT-11-10 15.5 (6.2) F11 C-HEGA-10 C-Hega

    -10 70 (3

    B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) F12 C-HEGA-11 C-Hega-11 23 (1

    C1 LPC-10 LysoPC-10 20 (8) G1 CYMAL-3 CYMAL-3 60 (3

    C2 LPC-12 LysoPC-12 7 (0.7) G2 CYMAL-4 CYMAL-4 19 (7

    C3 FOSFEN-9 FOSFEN-9 4.05 (1.35) G3 CYMAL-5 CYMAL-5 7.2 (2

    C4 CHAPS CHAPS 20 (8) G4 CYMAL-6 CYMAL-6 5.6 (0

    C5 CHAPSO CHAPSO 20 (8) G5 CYMAL-7 CYMAL-7 9.5 (0

    C6 DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) G6 DMHM 2,6-Dimethyl-4-heptyl--D-maltoside 55 (2

    C7 DDMAB n-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) G7 OM n-Octyl--D-maltopyranoside 39 (1

    C8 LAPAO LAPAO 4.8 (1.6) G8 NM n-Nonyl--D-maltopyranoside 15 (

    C9 TRIPAO TRIPAO 13.5 (4.5) G9 DM n-Decyl--D-maltopyranoside 4.8 (

    C10 T-20 TWEEN

    20 5.9 (0.059) G10 DM n-Decyl--D-maltopyranoside 5.4 (

    C11 BRIJ-35 BRIJ35 9.1 (0.091) G11 UDM n-Undecyl--D-maltopyranoside 5.8 (0

    C12 TX-100 TRITON

    X-100 11.5 (0.23) G12 UDM n-Undecyl--D-maltopyranoside 5.9 (0

    D1 TX-114 TRITON

    X-114 10 (0.2) H1 UDM -Undecylenyl--D-maltopyranoside 3.6 (

    D2 TX-305 TRITON

    X-305 6.5 (0.65) H2 DDM n-Dodecyl--D-maltopyranoside 7.5 (0

    D3 TX-405 TRITON

    X-405 8.1 (0.81) H3 DDM n-Dodecyl--D-maltopyranoside 8.5 (0

    D4 NID-P40 [Octylphenoxy]polyethoxyethanol 15 (0.3) H4 TDM n-Tridecyl--D-maltopyranoside 1.5 (0

    D5 APO8 Dimethyloctylphosphine oxide 80 (40) H5 OTM n-Octyl--D-thiomaltopyranoside 21.25

    D6 APO9 Dimethylnonylphosphine oxide 20 (10) H6 NTM n-Nonyl--D-thiomaltopyranoside 9.6 (3

    D7 APO10 Dimethyldecylphosphine oxide 13.98 (4.66) H7 DTM n-Decyl--D-thiomaltopyranoside 9 (0

    D8 APO11 Dimethylundecylphosphine oxide 3.6 (1.2) H8 UDTM n-Undecyl--D-thiomaltopyranoside 10.5 (0

    D9 APO12 Dimethyldodecylphosphine oxide 5.7 (0.57) H9 DDTM n-Dodecyl--D-thiomaltopyranoside 5 (0.

    D10 C6E3 Triethylene glycol monohexyl ether 46 (23) H10 S-8 Sucrose8 48.8 (2

    D11 C6E4 Tetraethylene glycol monohexyl ether 60 (30) H11 S-10 Sucrose10 7.5 (2

    D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) H12 S-12 Sucrose12 15 (0

    le1

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    The components in the detergent plate are shown above along with their locations in the plate

    and concentrations used. The values in parenthesis in the [Det] column are the CMC values for

    each detergent. Detergents in bold were purchased from Avanti Polar Lipids, italics from

    Bachem, underlined from EMD Biosciences, and all others from Anatrace. Detergents A3

    through C9 are zwitterionic while C10 through H12 are nonionic detergents.